To complement the work being done in 3D in vitro models using small molecule inhibitor platforms, the researchers have developed protocols to reverse transfect 3D layered and organoid models in an effort to understand the effect of specific gene perturbations using RNAi and CRISPR reagents, on morphology, survival and their relevance to small molecule inhibition. Described here is the development of an HTS compatible 3D spheroid based screening platform based on 384-well round bottom ULA plates. The platform generates one spheroid per well with size of approximately 400 micrometer in diameter and includes multiple assay readouts of tumor viability, including size, cell viability and cell death over time. The real time study of the effect of gene knockdown on proliferation and morphology of 3D spheroids adds a temporal component which we hope will better replicate pharmacological effects observed in tumors in vivo, including on set and duration of efficacy and resistance. The development of 3D physiologically relevant models to study therapeutic effect and the stromal contribution to acquisition of drug resistance and metastasis is extremely critical in the understanding of tumor biology and the subsequent design of therapeutic regimens for patients.